Larval black flies in the Simulium (Eusimulium) aureum Fries species group from the Greek island of Rhodes were chromosomally analyzed to clarify species limits and relationships with mainland taxa. Three species were identified, corresponding with earlier morphological identifications: S. flexibranchium Crosskey, S. petricolum (Rivosecchi), and S. rubzovianum (Sherban). The morphologically established endemic species S. flexibranchium shows chromosomal banding equivalency with S. angustipes Edwards, a widespread Palearctic species; the pair, therefore, are homosequential species, the ninth such example in the Simuliidae. The species status of S. flexibranchium is maintained on the basis of structural characters that allow diagnosis across the four islands from which it is known in the Aegean Sea. The chromosomal banding sequences of Rhodian populations of S. petricolum and S. rubzovianum, as well as a population of the latter that we examined from Cyprus, conform to the classic sequences of these species across their ranges, but tend toward the monomorphic condition typical of island black flies. The first definitive hybrid in the S. aureum group, a larva of S. flexibranchium × S. petricolum, was discovered chromosomally, and suggests an overall hybridization rate in the S. aureum group, with hybrid survival to the late larval stage, of less than 0.1%.